We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting
protein 2/
d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of
laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and
laser-microdissected from three different brain regions of two patients with
Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE
gels after boiling in Laemmli's sample
buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or
detergent, including 6 M
guanidine hydrochloride, 8 M
urea, and 2% SDS, the
laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under
biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total
protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of
proteins possessing an extensive aggregation property, particularly in small amounts.